There is a growing demand for binding molecules consisting of amino acids which are not immunoglobulins. While until now antibodies represent the best-established class of binding molecules there is still a need for new binding molecules in order to target ligands with high affinity and specificity since immunoglobulin molecules suffer from major drawbacks. Although they can be produced quite easily and may be directed to almost any target, they have a quite complex molecular structure. There is an ongoing need to substitute antibodies by smaller molecules which can be handled in an easy way. These alternative binding agents can be beneficially used for instance in the medical fields of diagnosis, prophylaxis and treatment of diseases.
Proteins having relatively defined 3-dimensional structures, commonly referred to as protein scaffolds, may be used as starting material for the design of said alternative binding agents. These scaffolds typically contain one or more regions which are amenable to specific or random sequence variation, and such sequence randomisation is often carried out to produce a library of proteins from which the specific binding molecules may be selected. Molecules with a smaller size than antibodies and a comparable or even better affinity towards a target antigen are expected to be superior to antibodies in terms of pharmacokinetic properties and immunogenicity.
For example, WO 04/106368 (Scil Proteins GmbH) describes the generation of artificial binding proteins on the basis of ubiquitin. Ubiquitin is a small, monomeric, and cytosolic protein which is highly conserved among Eucaryota. In the organism, it plays a crucial role in an enormous range of physiological processes and attachment of ubiquitin is the second-most common posttranslational modification following phosphorylation.
Ubiquitin is particularly characterized by beta sheets arranged in an antiparallel manner and subdivided into α and β segments. A characteristic of ubiquitin protein is an antiparallel beta sheet exposed to one surface of the protein onto the back side of which a α helix is packed which lies perpendicularly on top of it. This ubiquitin-like folding motif clearly distinguishes ubiquitin from other proteins.